The heptavalent pneumococcal conjugate vaccine (PCV7) has been shown to reduce the incidence of acute otitis media (AOM) caused by Streptococcus pneumoniae by 34% and reduces the overall incidence of AOM by 6% to 8%. More recent studies have shown increases in the proportion of Haemophilus influenzae and Moraxella catarrhalis in the middle-ear fluid of PCV7-immunized children. There has been no report on the effect of PCV7 on all 3 bacterial pathogens combined, either in the middle-ear fluid or nasopharynx of individual children with AOM. We investigated the impact of PCV7 on nasopharyngeal colonization with bacterial pathogens during AOM in the pre-PCV7 and post-PCV7 vaccination eras. Four hundred seventeen children (6 months to 4 years of age) were enrolled onto AOM studies between September 1995 and December 2004. Of these, 200 were enrolled before the vaccine use (historical controls), and 217 were enrolled after the initiation of PCV7 vaccination (101 were underimmunized, and 116 were immunized). Although the nasopharyngeal colonization rate for S pneumoniae was not different between the 3 groups, a significantly higher proportion of PCV7-immunized children with AOM were colonized with M catarrhalis. Overall, the mean number of pathogenic bacteria types isolated from immunized children (1.7) was significantly higher than in controls (1.4). The increase in bacterial colonization of the nasopharynx during AOM could be associated with an increase in AOM pathogens and theoretically can predispose PCV7-immunized children with AOM to a higher rate of antibiotic treatment failure or recurrent AOM.
Acute otitis media (AOM) is a very common childhood disease, and Streptococcus pneumoniae is one of the most common bacterial pathogens that cause AOM.1 The heptavalent pneumococcal conjugate vaccine (PCV7), directed against the 7 most common pneumococcal serotypes that cause invasive diseases, was licensed in February of 2000.2 The vaccine includes the 5 most common serotypes (19F, 6B, 23F, 14, and 9V) found in the middle-ear fluid (MEF) of children with AOM. Taking into account the vaccine serotypes and the serotypes that PCV7 may cross-protect, the vaccine has a potential coverage for 57% to 85% of AOM serotypes.3,4 Prelicensure studies have shown that in children vaccinated in the first year of life, PCV7 reduces pneumococcal AOM by 34% and the overall AOM incidence by 6% to 8%.5,6 Recent studies on the effect of PCV7 on AOM have shown a significant reduction in the incidence of AOM and alteration of the microbiology of AOM.7–9
The effect of PCV7 on reduction of AOM incidence is presumed to be caused by the reduction in colonization of vaccine serotypes of S pneumoniae in the nasopharynx of children. However, because of increased colonization of nonvaccine serotypes (serotype replacement), the overall nasopharyngeal colonization rate of S pneumoniae has not changed significantly.10,11 The studies on the effect of PCV7 on nasopharyngeal colonization have been performed only in children during the healthy period. During AOM episodes, there is a significant change in the microenvironment of the nasopharynx, including an increase in nasopharyngeal carriage of pathogenic bacteria, and a decline in nonpathogenic resident flora.12 To date, there has been no study on the effect of PCV7 on nasopharyngeal colonization of all AOM pathogens at the time of AOM development. For this study we evaluated 417 infants and children enrolled in clinical AOM trials at the University of Texas Medical Branch (UTMB) clinics during an 8-year period before and after the licensure of PCV7. The objective of the study was to evaluate the effect of PCV7 on nasopharyngeal bacterial colonization during AOM episodes. We also analyzed the changes over time on the proportion of AOM cases with colonized S pneumoniae and the susceptibility to penicillin.
This is a secondary data analysis from 4 independent AOM studies conducted at UTMB Galveston between October 1995 and December 2004. The studies were approved by the institutional review board. Informed consent was obtained from the legal guardians of all children. Of 580 children with AOM (3 months to 12 years of age) enrolled onto 4 studies (refs 13–15 and T.C., unpublished data), 417 cases fit the inclusion criteria for this analysis: (1) age between 6 months and 4 years (peak age group for AOM and eligibility for PCV7 vaccine); (2) no antibiotic in the previous week; and (3) nasopharyngeal bacterial culture obtained at study enrollment.
Of the 4 AOM studies from which the 417 cases were derived, studies 113 and 214 were performed before and studies 315 and 4 were performed after the initiation of PCV7 vaccine use at the UTMB pediatric clinics in August 2000. Children (3 months to 12 years) were generally healthy and received no antibiotics in the 7 days before enrollment except for 4 subjects in study 3, who were excluded from this analysis. Of the 4 patients excluded because of previous antibiotic use, 3 were underimmunized and 1 was fully immunized. The diagnosis of AOM was made by 1 of the experienced UTMB otitis researchers (T.C., D.P.M., K.S., or K.R.) on the basis of symptoms of fever, irritability, or earache, signs of inflammation of the tympanic membrane (red or yellow color or bulging of the membrane), and the presence of fluid in the middle ear as documented by pneumatic otoscopy or tympanometry. Tympanocentesis was performed only in study 1 (1995–1998).13 All studies were performed throughout the year. At enrollment and/or the time of AOM diagnosis, demographic and risk-factor information was collected. This information included previous number of ear infections, time in day care (hours per week), breastfeeding duration (weeks), number of children in the household, and cigarette smoke exposure. The children received treatment for AOM and returned for follow-up visits according to each study protocol. Children in studies 1 and 2 (3 months to 6 years of age) were treated with intramuscular ceftriaxone and randomly assigned to receive antihistamine, corticosteroid, both drugs, or placebo. Children in study 3 (6 months to 12 years of age) were randomly assigned to the watchful waiting or antibiotic groups. Children in study 4 (6 months to 4 years of age) were in a longitudinal study of AOM development after upper respiratory tract infection; AOM was treated with either antibiotics or watchful waiting. The number of cases included in the present analysis (on the basis of fulfillment of the above-mentioned age and culture-availability criteria) and the number of cases originally enrolled onto each study were 64 of 80 (study 1), 136 of 179 (study 2), 169 of 223 (study 3), and 48 of 98 (study 4).
Patient Groups and Definitions
A total of 417 children were divided into 3 groups on the basis of the time of enrollment and immunization status. Initiation of PCV7 at the UTMB clinics was in August 2000. Two hundred cases were designated as historical controls (cases enrolled before August 2000), and 217 children were enrolled after PCV7 availability (101 were underimmunized, and 116 were immunized fully or satisfactorily). Children were considered satisfactorily immunized when they had received all age-appropriate doses of PCV7 according to the Advisory Committee on Immunization Practices (ACIP) immunization schedule16 or the Centers for Disease Control and Prevention recommended shortage schedule.17 Of 116 immunized patients, 53 were fully immunized for age according to the ACIP schedule16 (eg, had received 3 doses of PCV7 if they were between 6 and 14 months, 4 doses of PCV7 if they were between 15 months and 4 years, or 1 dose of PCV7 after 24 months of age). Sixty-three children were immunized according to the Centers for Disease Control and Prevention recommended shortage schedule17: 36 were 6 to 14 months old and had received 2 doses of PCV7; 16 were 15 to 24 months old and had received 3 doses; 3 were 15 to 24 months old and had received 2 doses before 12 months of age; 7 were >24 months old and had received 2 doses before 12 months of age; and 1 was 19 months old and had received 2 doses, 1 before and 1 after 12 months. Children were considered underimmunized if they had not received any vaccine or received only 1 dose before the age of 24 months.
Specimen Collection and Processing
Nasopharyngeal samples for bacterial cultures were collected at enrollment by using Mini-Tip Culturette kits (Becton Dickinson Microbiology Systems, Cockeysville, MD). The specimens were submitted for routine bacterial cultures on blood and chocolate agar plates. Isolates of S pneumoniae were identified by using the optochin disk–susceptibility test (Taxo P; Becton Dickinson Microbiology Systems); S pneumoniae isolates were not serotyped. Isolates of Moraxella catarrhalis were identified by using the API QuadFerm assay (bioMerieux, Inc, Hazelwood, MO), and isolates of nontypeable H influenzae were identified by the Haemophilus ID Quad Plate with growth factors (Becton Dickinson Microbiology Systems).
The susceptibility of S pneumoniae to penicillin was determined by disk diffusion with a 1-μg oxacillin disk (Becton Dickinson Microbiology Systems) on Mueller Hinton agar with 5% sheep blood (Becton Dickinson Microbiology Systems). Isolates with a zone of inhibition of ≥20 mm were considered susceptible to penicillin. For isolates with a zone of inhibition of ≤19 mm, additional testing by the E test (AB Biodisk, Piscataway, NJ) was performed to determine the minimal inhibitory concentration for penicillin (susceptible, ≤0.06 μg/mL; intermediate, 0.12–1.0 μg/mL; resistant, ≥2 μg/mL). The susceptibility of S pneumoniae to ceftriaxone was also determined by the E test (susceptible, ≤0.5 μg/mL; intermediate, 1.0 μg/mL; resistant, ≥2.0 μg/mL). M catarrhalis and nontypeable H influenzae were tested for β-lactamase production by using the cefinase disk test (Becton Dickinson Microbiology Systems). Susceptibility testing was performed and interpreted according to the recommendations of the National Committee for Clinical Laboratory Standards.
Proportions were compared among groups by using the χ2 test or Fisher's exact test when assumptions of the χ2 test were not met. Means were compared by using analysis of variance. Trends were analyzed by using the 2-sided Cochran-Armitage trend test. Logistic-regression analysis was used for predicting the binary outcome of nasopharyngeal colonization (yes versus no) by using a set of potential risk factors. All analyses were conducted by using SAS statistical software (SAS Institute Inc, Cary, NC).
The median age of all children was 14 months (range: 6–48 months), 208 (50%) were male, 129 (31%) were white, 134 (32%) were black, 139 (33%) were Hispanic, and 15 (4%) were of other races (8 Asian and 7 of mixed races, predominately mixed white and black). One hundred eighty-six (45%) of the children attended day care. One hundred forty-six (35%) of the children were exposed to secondhand smoke, and 163 (39%) were breastfed. Of those children who were breastfed, the mean duration of breastfeeding was 9 weeks. Demographic and risk-factor data of children in 3 different groups are compared in Table 1. Overall, 76% of the children were <2 years old (79% in the historical control group, 66% in the underimmunized group, and 78% in the immunized group); there was no significant difference in age between groups.
At the time of AOM diagnosis, 84% of the historical control group, 91% of the underimmunized group, and 93% of the immunized group had pathogenic bacteria isolated from nasopharyngeal culture. S pneumoniae was isolated from nasopharyngeal samples from 208 children, nontypeable H influenzae was isolated from 147 (35%), and M catarrhalis was isolated from 261 (63%). Streptococcus pyogenes was isolated from 4 (1%) children; it was the only potential pathogen isolated in 1 of the 4 children. Samples from 175 (42%) of the children grew 2 AOM pathogens, and 38 (9%) grew 3 pathogens. Samples from 50 (12%) of the children did not grow any of these pathogens.
Figure 1 illustrates the proportion of nasopharyngeal bacterial colonization by each of the 3 major pathogens according to the groups of children. S pneumoniae colonization was similar in all groups: 48% of historical controls, 53% of the underimmunized group, and 51% of the immunized group were colonized with S pneumoniae (P = .60). A greater proportion of samples from the children in the immunized group grew nontypeable H influenzae (33% in historical controls, 32% in underimmunized children, and 42% in immunized children); the difference was not statistically significant (P = .17). A higher proportion of samples from immunized children (74%) were colonized with M catarrhalis, compared with those from the historical controls and underimmunized groups (56% and 62%, respectively) (P = .006). A larger percentage of samples from children in the historical control group did not grow any nasopharyngeal pathogen (16%) compared with those from the underimmunized and immunized groups (9% and 7%, respectively) (P = .033). The average number of types of pathogenic bacteria in the nasopharynx per episode of AOM is shown in Table 2. The mean number of types of bacteria was 1.37 (SD: ±0.83) in the historical control group, 1.48 (SD: ±0.79) in the underimmunized group, and 1.67 in the immunized group (SD: ±0.79) (P = .006 by analysis of variance). Pairwise comparison of the groups showed a difference in the mean number of bacteria types in the immunized group compared with the historical controls (P < .05).
To compare the full effect of PCV7 on nasopharyngeal colonization of S pneumoniae during AOM, we analyzed a subset of 98 children from the post-PCV7 era who were either fully immunized with PCV7 according to the original ACIP immunization schedule (n = 53) or were <2 years of age and had not received any doses of PCV7 (n = 45). The results were similar to those shown in Fig 1. However, because of the small number, statistical significance was not achieved between these 2 groups with respect to nasopharyngeal colonization with S pneumoniae (53% in the fully immunized vs 58% in the no-vaccination group; P = .6), nontypeable H influenzae (43% vs 33%; P = .3), or M catarrhalis (68% vs 58%; P = .3). We also compared the proportion of nasopharyngeal colonization with S pneumoniae in children fully immunized based on the ACIP schedule (n = 53) to those who missed 1 (n = 52) or 2 (n = 11) PCV7 doses as a result of the shortage. The rate of nasopharyngeal colonization with S pneumoniae in children who were fully immunized was 53%, compared with 50% in children who missed 1 PCV7 dose and 45% in children who missed 2 doses of PCV7 from the vaccine shortage. There was no difference in the nasopharyngeal colonization rate of S pneumoniae between fully immunized children and in children missing 1 or 2 doses (P = .8 and 0.7, respectively).
Penicillin-Resistant S pneumoniae
Table 3 shows the proportion of AOM case subjects with S pneumoniae in the nasopharynx and the changes in S pneumoniae–resistant patterns from September 1995 through December 2004 according to group and year. The proportion of samples from AOM case subjects colonized with S pneumoniae was stable throughout the study period. There was no difference in the overall proportion of penicillin-resistant strains of S pneumoniae (PR-Sp) between the 3 groups throughout the study years (historical controls, 15%; underimmunized, 15%; immunized, 12%). Since the introduction of PCV7, however, the proportion of PR-Sp isolates decreased significantly with each passing year in both the underimmunized and immunized groups (both P < .03 by 2-sided Cochran-Armitage trend test).
Nontypeable H influenzae and M catarrhalis
The proportion of nontypeable H influenzae and M catarrhalis isolated from the nasopharynx of AOM case subjects according to year from September 1995 through December 2004 ranged from 29% to 47% and 40% to 80%, respectively (data not shown). There was a trend for the overall proportion of isolates of both bacteria types to be increased with the use of PCV7 since the year 2000 (P = .08 by 2-sided Cochran-Armitage trend test). The proportion of the isolates that were β-lactamase-positive nontypeable H influenzae remained stable over the years (P = .6 by 2-sided Cochran-Armitage trend test).
We ran a logistic-regression model to control for the effect of day care, breastfeeding, previous otitis media, and smoke exposure on nasopharyngeal colonization among the groups. The differences for nasopharyngeal colonization with S pneumoniae and nontypeable H influenzae were not significant (P = .56 and .12, respectively). The difference for M catarrhalis colonization remained significant (P = .04).
In the present study, we found that in children with AOM, immunization with PCV7 was not associated with reduction in S pneumoniae colonization in the nasopharynx. As has been reported in the MEF,4,7,8M catarrhalis and nontypeable H influenzae isolation from the nasopharynx of our PCV7-immunized children with AOM increased. Our finding of increased overall nasopharyngeal colonization with more types of pathogenic bacteria during AOM is novel and provocative. The possible negative effect of PCV7 vaccination on nasopharyngeal colonization in children who subsequently develop AOM has not been previously reported.
We did not perform serotyping of S pneumoniae isolates in our study. It is likely, however, that serotype replacement was the underlying reason for the stable rate of nasopharyngeal colonization with S pneumoniae at AOM diagnosis during the pre-PCV7 and post-PCV7 eras. The serotype-replacement phenomenon has been observed in many recent studies, including the studies on the effect of PCV7 on nasopharyngeal colonization of S pneumoniae in healthy young infants and children during the first few years of life11,18 and in a study of children >1 year of age with history of recurrent AOM who received both PCV7 and 23-valent pneumococcal polysaccharide vaccine.10 In a study of the MEF of PCV7-vaccinated children with AOM, Eskola et al5 also found a 33% increase in the rate of AOM attributed to pneumococcal serotypes not included in the vaccine. In a multisite study, McEllistrem et al19 found an increased proportion of non-PCV7 S pneumoniae serogroups in the MEF of children with AOM. Our finding of reduction in PR-Sp strains in the nasopharynx during AOM over the years after PCV7 use also supports this notion.
Studies of the MEF from children with AOM have shown the increased proportion of H influenzae and M catarrhalis in PCV7-immunized children.5,7,8 There has been no report, to date, on the effect of PCV7 on the overall bacteriologic data combining 3 common pathogenic bacteria in the MEF or nasopharynx during the individual AOM episode. Why PCV7-immunized children were colonized with more pathogenic bacteria other than S pneumoniae was unclear. It was possible that the vaccine effect on elimination of vaccine serotypes of S pneumoniae interfered with the natural balance of microbial species in the nasopharynx. Earlier studies have shown bacterial interference phenomenon between normal flora and pathogenic bacteria.20,21 More recent studies have shown an increased incidence of Staphylococcus aureus–related otitis media after PCV7 vaccination10 and a negative correlation for nasopharyngeal colonization of vaccine-type S pneumoniae and S aureus in healthy children,22 which suggests natural competition between colonization with the 2 bacteria.
We were able to include a large number of children with AOM, diagnosed before licensure of PCV7, as historical controls in our study. Although our historical controls differed from the children in the PCV7 era with respect to the rate of breastfeeding and smoke exposure, these differences may reflect cultural changes over time toward less smoking and more breastfeeding. The fact that controls had a lower rate of breastfeeding and higher rate of smoke exposure lends even more support to our study results, because these factors should have been associated with an increased rate of nasopharyngeal colonization with pathogenic bacteria, not a reduction.23
The periodic shortage of the vaccine in the first few years after licensure had limited the efforts to fully immunize children with PCV7 in our area, resulting in a sizable group of underimmunized children. The data presented here, therefore, are derived from the children with AOM for whom PCV7 vaccination status was classified to be immunized or underimmunized on the basis of the shortage schedule.17 Nevertheless, when only a subset of children who were completely immunized was compared with a subgroup of children <24 months of age in the post-PCV7 cohort who had not received any PCV7, the trend for nasopharyngeal colonization with all 3 bacteria remained the same. Furthermore, we did not find a difference in the S pneumoniae colonization rate between children with AOM who received all recommended PCV7 doses and those who missed 1 or 2 doses of PCV7 because of vaccine shortage. The current recommendation of routine PCV7 immunization will preclude any future study aiming to compare the data from completely immunized children to those who have received no PCV7 vaccine.
Because our study specifically included children who had already developed AOM, we could not assess the effect of PCV7 on the overall reduction of AOM incidence. In addition, we studied nasopharyngeal colonization patterns instead of MEF cultures. It is believed that AOM pathogens derive from bacteria colonized in the nasopharynx when events such as viral respiratory infection enhance adherence and colonization of nasopharyngeal bacterial flora and alter eustachian tube function.24,25 The correlation between pathogens found in the nasopharynx and in the middle ear has been studied.12,26,27 Although the positive predictive value of nasopharyngeal cultures has been found to be as low as 0.50 to 0.71 for nontypeable H influenzae, 0.22 to 0.44 for S pneumoniae, and 0.17 to 0.19 for M catarrhalis, negative predictive values were high, ranging from 95% to 99%. There is a close agreement between MEF and nasopharyngeal culture data for nontypeable H influenzae and S pneumoniae, especially with regard to resistance patterns. Epidemiologically, nasopharyngeal culture is an acceptable alternative to MEF culture for the monitoring of resistance patterns.27
The clinical significance of increased nasopharyngeal colonization of pathogenic bacteria, especially M catarrhalis in PCV7-immunized children who develop AOM, remains to be seen. Some studies have shown the effect of PCV7 in reducing recurrent AOM.7,28 On the other hand, Libson et al29 reported an association between nasopharyngeal carriage of S pneumoniae at the completion of successful antibiotic treatment of AOM with predisposition to early clinical recurrence. Theoretically, polymicrobial nasopharyngeal colonization with pathogenic bacteria during AOM could increase the likelihood of polymicrobial AOM. This could possibly lead to a higher rate of failure to respond to single-antibiotic treatment or increased risk for development of recurrent AOM after antibiotic treatment. Our results suggest the need for continued vigilance on the effect of PCV7 on AOM.
This work was supported by National Institutes of Health grants R01 DC 2620, R01 DC 5841, and R01 HS 10613 from the Agency for Healthcare Research and Quality. The study was conducted at the General Clinical Research Center at the University of Texas Medical Branch, funded by grant M01 RR 00073 from the National Center for Research Resources, National Institutes of Health, US Public Health Service.
We thank LiFang Zhang, MS, for assistance with data preparation and analysis; Stephen I. Pelton, MD, for critical review of the manuscript; M. Lizette Rangel, Kyralessa B. Ramirez, Syed Ahmad, Liliana Najera, and Michelle Tran for assistance with study subjects; and Sangeeta Nair and Rafael Serna for assistance in the laboratories.
- Accepted October 13, 2005.
- Address correspondence to Tasnee Chonmaitree, MD, Department of Pediatrics, Division of Infectious Diseases, University of Texas Medical Branch, 301 University Blvd, Galveston, TS 77555-0371. E-mail:
The authors have indicated they have no financial relationships relevant to this article to disclose.
This work was presented in part at the 5th Extraordinary International Symposium on Recent Advances in Otitis Media; April 24–27, 2005; Amsterdam, Netherlands; and the Pediatric Academic Societies' meeting; May 14–17, 2005; Washington, DC.
- ↵American Academy of Pediatrics, Committee on Infectious Diseases. Policy statement: recommendations for the prevention of pneumococcal infections, including the use of pneumococcal conjugate vaccine (Prevnar), pneumococcal polysaccharide vaccine, and antibiotic prophylaxis. Pediatrics.2000;106 :362– 366
- ↵Talbot TR, Poehling KA, Hartert TV, et al. Reduction in high rates of antibiotic-nonsusceptible invasive pneumococcal disease in Tennessee after introduction of the pneumococcal conjugate vaccine. Clin Infect Dis.2004;39 :641– 648
- ↵Ghaffar F, Barton T, Lozano J, et al. Effect of the 7-valent pneumococcal conjugate vaccine on nasopharyngeal colonization by Streptococcus pneumoniae in the first 2 years of life. Clin Infect Dis.2004;39 :930– 938
- ↵McCormick DP, Chonmaitree T, Pittman C, et al. Non-severe acute otitis media: a clinical trial comparing outcomes of watchful waiting versus immediate antibiotic treatment. Pediatrics.2005;115 :1455– 1465
- ↵American Academy of Pediatrics, Committee on Infectious Diseases. Recommended childhood and adolescent immunization schedule: United States, July–December 2004. Pediatrics.2004;113 :1448
- ↵McEllistrem MC, Adams JM, Patel K, et al. Acute otitis media due to penicillin-nonsusceptible Streptococcus pneumoniae before and after the introduction of the pneumococcal conjugate vaccine. Clin Infect Dis.2005;40 :1738– 1744
- ↵Sanders CC, Sanders WE Jr, Harrowe DJ. Bacterial interference: effects of oral antibiotics on the normal throat flora and its ability to interfere with group A streptococci. Infect Immun.1976;13 :808– 812
- ↵Duffy LC, Faden H, Wasielewski R, Wolf J, Krystofik D. Exclusive breastfeeding protects against bacterial colonization and day care exposure to otitis media. Pediatrics.1997;100(4) . Available at: www.pediatrics.org/cgi/content/full/100/4/e7
- ↵Bluestone CD, Klein JO. Physiology, pathophysiology and pathogenesis. In: Bluestone CD, Klein JO, eds. Otitis Media in Infants and Children. Philadelphia, PA: WB Saunders; 1995:17–37
- ↵Howie VM, Ploussard JH. Simultaneous nasopharyngeal and middle ear exudate culture in otitis media. Pediatr Dig.1971;13 :31– 35
- ↵Libson S, Dagan R, Greenberg D, et al. Nasopharyngeal carriage of Streptococcus pneumoniae at the completion of successful antibiotic treatment of acute otitis media predisposes to early clinical recurrence. J Infect Dis.2005;191 :1869– 1875
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